Peptide fusion improves prime editing efficiency.

Prime editing enables search-and-replace genome editing but is limited by low editing efficiency. We present a high-throughput approach, the Peptide Self-Editing sequencing assay (PepSEq), to measure how fusion of 12,000 85-amino acid peptides influences prime editing efficiency. We show that peptide fusion can enhance prime editing, prime-enhancing peptides combine productively, and a top dual peptide-prime editor increases prime editing significantly in multiple cell lines across dozens of target sites. Top prime-enhancing peptides function by increasing translation efficiency and serve as broadly useful tools to improve prime editing efficiency.

Comprehensive Mapping of Key Regulatory Networks that Drive Oncogene Expression.

Gene expression is controlled by the collective binding of transcription factors to cis-regulatory regions. Deciphering gene-centered regulatory networks is vital to understanding and controlling gene misexpression in human disease; however, systematic approaches to uncovering regulatory networks have been lacking. Here we present high-throughput interrogation of gene-centered activation networks (HIGAN), a pipeline that employs a suite of multifaceted genomic approaches to connect upstream signaling inputs, trans-acting TFs, and cis-regulatory elements. We apply HIGAN to understand the aberrant activation of the cytidine deaminase APOBEC3B, an intrinsic source of cancer hypermutation. We reveal that nuclear factor κB (NF-κB) and AP-1 pathways are the most salient trans-acting inputs, with minor roles for other inflammatory pathways. We identify a cis-regulatory architecture dominated by a major intronic enhancer that requires coordinated NF-κB and AP-1 activity with secondary inputs from distal regulatory regions. Our data demonstrate how integration of cis and trans genomic screening platforms provides a paradigm for building gene-centered regulatory networks.

Elucidation of remdesivir cytotoxicity pathways through genome-wide CRISPR-Cas9 screening and transcriptomics.

The adenosine analogue remdesivir has emerged as a front-line antiviral treatment for SARS-CoV-2, with preliminary evidence that it reduces the duration and severity of illness1.Prior clinical studies have identified adverse events1,2, and remdesivir has been shown to inhibit mitochondrial RNA polymerase in biochemical experiments7, yet little is known about the specific genetic pathways involved in cellular remdesivir metabolism and cytotoxicity. Through genome-wide CRISPR-Cas9 screening and RNA sequencing, we show that remdesivir treatment leads to a repression of mitochondrial respiratory activity, and we identify five genes whose loss significantly reduces remdesivir cytotoxicity. In particular, we show that loss of the mitochondrial nucleoside transporter SLC29A3 mitigates remdesivir toxicity without a commensurate decrease in SARS-CoV-2 antiviral potency and that the mitochondrial adenylate kinase AK2 is a remdesivir kinase required for remdesivir efficacy and toxicity. This work elucidates the cellular mechanisms of remdesivir metabolism and provides a candidate gene target to reduce remdesivir cytotoxicity.

A CRISPR view of gene regulation.

Due to plummeting costs, whole genome sequencing of patients and cancers will soon become routine medical practice; however, we cannot currently predict how non-coding genotype affects cellular gene expression. Gene regulation research has recently been dominated by observational approaches that correlate chromatin state with regulatory function. These approaches are limited to the available genotypes and cannot scratch the surface of possible sequence combinations, and thus there is a need for perturbation-based approaches to better understand how DNA encodes gene regulatory functions. CRISPR/Cas9 genome editing has revolutionized our ability to alter genome sequence, and CRISPR/Cas9-based assays have already begun to contribute to new paradigms of gene regulation. We discuss the variety of arenas in which current and future CRISPR-based technologies will aid in developing predictive understanding of how genome sequence leads to gene regulatory function.

A CD47-associated super-enhancer links pro-inflammatory signalling to CD47 upregulation in breast cancer.

CD47 is a cell surface molecule that inhibits phagocytosis of cells that express it by binding to its receptor, SIRPα, on macrophages and other immune cells. CD47 is expressed at different levels by neoplastic and normal cells. Here, to reveal mechanisms by which different neoplastic cells generate this dominant 'don't eat me' signal, we analyse the CD47 regulatory genomic landscape. We identify two distinct super-enhancers (SEs) associated with CD47 in certain cancer cell types. We show that a set of active constituent enhancers, located within the two CD47 SEs, regulate CD47 expression in different cancer cell types and that disruption of CD47 SEs reduces CD47 gene expression. Finally we report that the TNF-NFKB1 signalling pathway directly regulates CD47 by interacting with a constituent enhancer located within a CD47-associated SE specific to breast cancer. These results suggest that cancers can evolve SE to drive CD47 overexpression to escape immune surveillance.

Self-Cloning CRISPR.

CRISPR/Cas9-gene editing has emerged as a revolutionary technology to easily modify specific genomic loci by designing complementary sgRNA sequences and introducing these into cells along with Cas9. Self-cloning CRISPR/Cas9 (scCRISPR) uses a self-cleaving palindromic sgRNA plasmid (sgPal) that recombines with short PCR-amplified site-specific sgRNA sequences within the target cell by homologous recombination to circumvent the process of sgRNA plasmid construction. Through this mechanism, scCRISPR enables gene editing within 2 hr once sgRNA oligos are available, with high efficiency equivalent to conventional sgRNA targeting: >90% gene knockout in both mouse and human embryonic stem cells and cancer cell lines. Furthermore, using PCR-based addition of short homology arms, we achieve efficient site-specific knock-in of transgenes such as GFP without traditional plasmid cloning or genome-integrated selection cassette (2% to 4% knock-in rate). The methods in this paper describe the most rapid and efficient means of CRISPR gene editing. © 2016 by John Wiley & Sons, Inc.

Gata6 potently initiates reprograming of pluripotent and differentiated cells to extraembryonic endoderm stem cells.

Transcription factor-mediated reprograming is a powerful method to study cell fate changes. In this study, we demonstrate that the transcription factor Gata6 can initiate reprograming of multiple cell types to induced extraembryonic endoderm stem (iXEN) cells. Intriguingly, Gata6 is sufficient to drive iXEN cells from mouse pluripotent cells and differentiated neural cells. Furthermore, GATA6 induction in human embryonic stem (hES) cells also down-regulates pluripotency gene expression and up-regulates extraembryonic endoderm (ExEn) genes, revealing a conserved function in mediating this cell fate switch. Profiling transcriptional changes following Gata6 induction in mES cells reveals step-wise pluripotency factor disengagement, with initial repression of Nanog and Esrrb, then Sox2, and finally Oct4, alongside step-wise activation of ExEn genes. Chromatin immunoprecipitation and subsequent high-throughput sequencing analysis shows Gata6 enrichment near pluripotency and endoderm genes, suggesting that Gata6 functions as both a direct repressor and activator. Together, this demonstrates that Gata6 is a versatile and potent reprograming factor that can act alone to drive a cell fate switch from diverse cell types.

Functional evaluation of ES cell-derived endodermal populations reveals differences between Nodal and Activin A-guided differentiation.

Embryonic stem (ES) cells hold great promise with respect to their potential to be differentiated into desired cell types. Of interest are organs derived from the definitive endoderm, such as the pancreas and liver, and animal studies have revealed an essential role for Nodal in development of the definitive endoderm. Activin A is a related TGFß member that acts through many of the same downstream signaling effectors as Nodal and is thought to mimic Nodal activity. Detailed characterization of ES cell-derived endodermal cell types by gene expression analysis in vitro and functional analysis in vivo reveal that, despite their similarity in gene expression, Nodal and Activin-derived endodermal cells exhibit a distinct difference in functional competence following transplantation into the developing mouse embryo. Pdx1-expressing cells arising from the respective endoderm populations exhibit extended differences in their competence to mature into insulin/c-peptide-expressing cells in vivo. Our findings underscore the importance of functional cell-type evaluation during stepwise differentiation of stem cells.

Conversion from mouse embryonic to extra-embryonic endoderm stem cells reveals distinct differentiation capacities of pluripotent stem cell states.

The inner cell mass of the mouse pre-implantation blastocyst comprises epiblast progenitor and primitive endoderm cells of which cognate embryonic (mESCs) or extra-embryonic (XEN) stem cell lines can be derived. Importantly, each stem cell type retains the defining properties and lineage restriction of their in vivo tissue of origin. Recently, we demonstrated that XEN-like cells arise within mESC cultures. This raises the possibility that mESCs can generate self-renewing XEN cells without the requirement for gene manipulation. We have developed a novel approach to convert mESCs to XEN cells (cXEN) using growth factors. We confirm that the downregulation of the pluripotency transcription factor Nanog and the expression of primitive endoderm-associated genes Gata6, Gata4, Sox17 and Pdgfra are necessary for cXEN cell derivation. This approach highlights an important function for Fgf4 in cXEN cell derivation. Paracrine FGF signalling compensates for the loss of endogenous Fgf4, which is necessary to exit mESC self-renewal, but not for XEN cell maintenance. Our cXEN protocol also reveals that distinct pluripotent stem cells respond uniquely to differentiation promoting signals. cXEN cells can be derived from mESCs cultured with Erk and Gsk3 inhibitors (2i), and LIF, similar to conventional mESCs. However, we find that epiblast stem cells (EpiSCs) derived from the post-implantation embryo are refractory to cXEN cell establishment, consistent with the hypothesis that EpiSCs represent a pluripotent state distinct from mESCs. In all, these findings suggest that the potential of mESCs includes the capacity to give rise to both extra-embryonic and embryonic lineages.

Determinants of skeletal muscle contributions from circulating cells, bone marrow cells, and hematopoietic stem cells.

To investigate the factors that regulate incorporation into uninjured or damaged skeletal muscle of donor markers derived from unfractionated bone marrow (BM) cells or from highly purified c-kit+ Thy1.1lo Lin- Sca-1+ hematopoietic stem cells (HSCs), we evaluated myofiber chimerism of multiple muscle groups in irradiated and transplanted recipient mice and in unirradiated parabiotic animals. Uninjured panniculus carnosus, diaphragm, and abdominal muscles infrequently incorporated donor markers into myofibers in a subset of animals after either BM or HSC transplantation; however, acute muscle injury was essential to elicit contributions to triceps surae (TS) and tibialis anterior muscles. The low level of incorporation of donor marker-expressing myofibers could not be enhanced either by transplantation into newborn recipients or by induced migration of HSCs into the periphery. Analysis of muscle chimerism in unirradiated animals joined surgically by parabiosis revealed that contributions of circulating cells to myofibers in the TS were injury dependent and that at least some circulating cells with the potential to contribute to regenerating muscle derive from BM, suggesting that hematoablative preconditioning is not required for such contributions. In all cases tested, donor-derived myofibers expressed both donor-specific and host-specific markers, suggesting that they arise by low-level fusion into skeletal muscle of cells that can include the progeny of HSCs. It is not yet clear whether such events represent a normal myogenic pathway or a pathological response to muscle damage.

Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle.

Skeletal muscle regeneration in adults is thought to occur through the action of myogenic satellite cells located in close association with mature muscle fibers; however, these precursor cells have not been prospectively isolated, and recent studies have suggested that additional muscle progenitors, including cells of bone marrow or hematopoietic origin, may exist. To clarify the origin(s) of adult myogenic cells, we used phenotypic, morphological, and functional criteria to identify and prospectively isolate a subset of myofiber-associated cells capable at the single cell level of generating myogenic colonies at high frequency. Importantly, although muscle-engrafted cells from marrow and/or circulation localized to the same anatomic compartment as myogenic satellite cells and expressed some though not all satellite cell markers, they displayed no intrinsic myogenicity. Together, these studies describe the clonal isolation of functional adult myogenic progenitors and demonstrate that these cells do not arise from hematopoietic or other bone marrow or circulating precursors.

Little evidence for developmental plasticity of adult hematopoietic stem cells.

To rigorously test the in vivo cell fate specificity of bone marrow (BM) hematopoietic stem cells (HSCs), we generated chimeric animals by transplantation of a single green fluorescent protein (GFP)-marked HSC into lethally irradiated nontransgenic recipients. Single HSCs robustly reconstituted peripheral blood leukocytes in these animals, but did not contribute appreciably to nonhematopoietic tissues, including brain, kidney, gut, liver, and muscle. Similarly, in GFP+:GFP- parabiotic mice, we found substantial chimerism of hematopoietic but not nonhematopoietic cells. These data indicate that "transdifferentiation" of circulating HSCs and/or their progeny is an extremely rare event, if it occurs at all.